The concentration of intracellular free calcium (Ca.sup.2+) is a major determinant of smooth muscle contraction. The influx of Ca.sup.2+ through voltage-sensitive Ca.sup.2+ channels into cells is an important mechanism for increasing intracellular Ca.sup.2+ concentration. Voltage-sensitive L-type Ca.sup.2+ channels are opened when cells depolarize. In sum, depolarization of a smooth muscle cell correlates with its contraction via influx of calcium ions.
The tendency for potassium (K.sup.+) to exit out of a cell is responsible for the cell's resting membrane potential. The opening of K.sup.+ channels increases the net negative charge of a resting cell (or hyperpolarizes it), and thereby, tends to make it more difficult to depolarize. In fact, when K.sup.+ channels are opened, depolarized cells will repolarize. Thus, the opening of K.sup.+ channels counteracts cellular depolarization, and therefore, prevents or reverses smooth muscle contraction.
"K.sup.+ channel openers" are a chemically heterogeneous group of compounds that hyperpolarize cells and thereby decrease excitability and inhibit the influx of Ca.sup.2+ into cells. K.sup.+ channel openers have proven efficacy as antihypertensive agents (Edwards et al., 1990, Pharmac. Ther., 48:237), and are potentially useful in the treatment of diseases whose etiology is due to excessive membrane excitability or excessive intracellular calcium levels.
An intact tissue assay for binding activity of K.sup.+ channel openers has been reported. Bray et al., 1992, J. Biol. Chem., 267:11689, report the binding of a putative K.sup.+ channel opener in rings cut from rat aorta. Intact tissue assays involve the use of large numbers of live animals which have to be maintained and sacrificed each time such assays are performed. Such assays are, therefore, cumbersome.
Furthermore, intact tissue assays have large variability in results because of inherent problems in washing nonspecifically associated radioligand out of the test tissue. For example, the assay disclosed in Bray et al. exhibited high variability which necessitated the carrying out of measurements in quadruplicate.
Thus, there is a need in the art for more efficient assays for detecting compounds which modulate K.sup.+ channels. The present specification discloses for the first time, the specific binding of a K.sup.+ channel opener, (+)-N-(2-ethoxyphenyl)-N'-(1,2,2,-trimethylpropyl)-2-nitroethene-1,1-diami ne (hereinafter "CMPD-I"), to intact cells. The synthesis of the racemate (+/-) of this compound is disclosed in U.S. Pat. No. 4,567,188 issued to Niemers et al. (see FIG. 1). The preferred radioligand used in the present assays is the tritiated (+)enantiomer (hereinafter "[.sup.3 H]CMPD-I").